New members of the order Thermotogales were isolated from nonvolcanically heated geothermal environments, including oil fields and waters of the Great Artesian Basin of Australia, thereby extending their known habitats, previously recognized primarily as volcanic. The hyperthermophilic and thermophilic members of Thermotogales of volcanic origin, together with the recently described nonvolcanic species of this order and three new isolates described in this paper, were all found to produce L-alanine from glucose fermentation, in addition to acetate, lactate, CO2 and H2. L-alanine production from glucose is a trait in common with Pyrococcus furiosus and Thermococcus profundus. We propose that L-alanine production from sugar fermentation be regarded as an ancestral metabolic characteristic.